Allele selective enrichment for targeted profiling of rare cancer mutations via low-depth sequencing

Summary Cell free DNAcfDNAin peripheral blood contain tumor derived DNA and are promising noninvasive biomarkers for cancer therapy selection and recurrence monitoringHoweverthe high cost of current commercial next generation sequencingNGSpanels for profiling cancer mutations in cfDNA prevents them from being routinely usedFurthermorethe high sequencing depth and reads required means that cfDNA profiling is essentially only possible in specialized centralized reference laboratories and inaccessible to hospitals with lowerthroughput sequencing instrumentsHerewe propose to use allele selective enrichment sequencingASESto develop NGS panels that are capable of profiling rare cancer mutations in cfDNA with less thanvariant allele frequencyVAFusing low depth sequencingWe achieve this using allele specific toehold probes that deplete wildtype DNA sequences and or enrich for known mutationsincreasing the VAF of rare cancer mutations by roughlyfoldUnlike other hybridizationbased probestoehold probes discriminate single nucleotide variants based on molecular competition rather than melting temperature optimizationand thus is uniquely scalable to high multiplexingWe have obtained preliminary data on a NGS panel that includesampliconsand observed similar mutation sensitivity usingfewer NGS readsIn this Phase I SBIR applicationwe will first develop an ASES NGS panel for cfDNA profiling coveringgenes bearingfrequent mutations in non small cell lung cancer patientsSubsequentlywe will scale up to akb hypothesis free panel coveringgenes Narrative Noninvasive profiling of cancer mutations in cell free DNA can inform optimal target therapy selection and provide timely early warning of cancer recurrencebut is currently not economically feasible due to the high sequencing depths requiredWe have develop Allele Specific Enrichment SequencingASESin which rare cancer mutations can be reliably and economically analyzed using low depth sequencingIn this SBIR Phase I proposalwe will first show feasibility on agene lung cancer ASES panel targetingrecurrent mutationsand then demonstrate a hypothesis free ASES panel covering the full exon regions ofgeneskb